Prokaryotic Expression Of The Recombinant PTB Domain Of Rat Mint2 And Development Of Its Polyclonal Antibody

ObjectiveThe deposition of 3 -amyloid protein(Aβ) in the brain is believed to be one of the causes of Alzheimer's disease(AD),and Aβ is the proteolytic product of amyloid precursor protein(APP). The Mints is a family of molecular adaptors. And as the numbers of the family, the Mintl and Mint2 express specifically in neurons. Many researches indicate that Mints are the important material which regulate APP metablism and Aβ production. Thus, the Mints may play a key role in the onset and development of AD.The Mint2 gene was found in the course of researching the interaction between Mintl and APP. The direct interaction of Mint2 with the cytoplasmic domain of APP can suppress the production of A 3 40 but not Aβ 42. This suppression of A 3 40 generation can be abolished by dissociation of Mint2-APP complex resulting from association of XB51 with Mint2. In the other way, Aβ42 production will deduce following the PDZ2 domain of Mint2 binding to the 161-FAQ-163 motif in the RHD domain of monomeric NF- KB/P65. Reasonably, Mint2 have been believed to be a candidate target to control the progression of AD.To investigate the function of Mint2 PTB domain in APP metablism and signal transduction, the recombinant PTB domain of rat Mint2 was reconstructed in the present study. Polyclonal antibody against the PTB domain was developed after the prokaryotic expression of the recombinant PTB domain of rat Mint2 in E. Coli. The proteins and antibodies can be credible tools in the research of the PTB-mediated protein-proteininteractions.Methods1. Amplification of Mint2 PTB gene by PCR: The primer was designed according to thecDNA sequence of rat Mint2 PTB in Genbank with the assistant tool DNAstar, and slightlyimprove the reaction condition refering to molecule clone.2.Sequence identification: Identification of DNA Sequence was performed at the JiKangcompany.3. Preparation of the pGEX4T3-PTB recimbinant plasmind: the pGEX4T3 vector and amplifying segment were both double digested by BamHI and Xhol, and then were linked by T4 ligase.4. Amplification and identification of the pGEX4T3-PTB: The pGEX4T3-PTB recombinant plasmid was amplified by converting TGI, and then identifed by double digestion.5.Protein expression and purification:The expression of GST-PTB fusion protein was induced with IPTG in the converted BL21(pGEX4T3-PTB) by 15%SDS-PAGE, and analysis the fusion protein by electrophoresis documentation and analysis system. The production was purified by Glutathione-Sepharose 4B.6.Development and detection of antibody: The antiserum was obtained by immunizing the BALB/C mice with the purified GST-PTB, and purified after removing the antibody against GST with GST -Sepharose 4B.The titre and specificity of antibody were detected by ELIS A and Western blot. Results1. Electrophoresis result of PCR segment: There was a bright band near the Marker 500bp; 2.Sequence identification: The segment which was shown in the 79-591 bp of the sequence map was corresponding to the 1320-1832bp of rat Mint2 PTB domain cDAN; 3.1dentification of the pGEX4T3-PTB: The double digested production were two bands in the 1% agarose electrophoresis, one was near 500bp and the other was over 2000bp. 4.The result of protein expression and purification: Fusion protein were detected near 43000 by SDS-PAGE, of which about twenty percent was appeared in the soluble form.The fusion protein was up to 95% after being purified.5.The detection of antibody: The antibody titre reached 1 : 26 730 by ELISA. There was noimmunoreation band at GST antibody location detected by Western blot.ConclusionThis study constructed successfully recombinant plasmid pGEX4T3-PTB and finished the soluble expression of the GST-PTB fusion protein in E.Coli. BL21, then obtained the high-titered specific polyclonal antibody (up to 1 : 26 730) against rat Mint2 PTB domain by immunizing the BALB/C mouse with the protein. The protein and antibody can be credible tools for researching the immunoblot and immun...